CN118032996A - Method for measuring content of L-glutamine in sports nutrition food - Google Patents
Method for measuring content of L-glutamine in sports nutrition food Download PDFInfo
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- CN118032996A CN118032996A CN202410287803.3A CN202410287803A CN118032996A CN 118032996 A CN118032996 A CN 118032996A CN 202410287803 A CN202410287803 A CN 202410287803A CN 118032996 A CN118032996 A CN 118032996A
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- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 title claims abstract description 51
- 229930182816 L-glutamine Natural products 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 29
- 235000013305 food Nutrition 0.000 title claims abstract description 22
- 235000014268 sports nutrition Nutrition 0.000 title abstract description 11
- 239000011550 stock solution Substances 0.000 claims abstract description 17
- 239000012224 working solution Substances 0.000 claims abstract description 17
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 238000004458 analytical method Methods 0.000 claims abstract description 5
- 239000012488 sample solution Substances 0.000 claims abstract 2
- 239000000243 solution Substances 0.000 claims description 30
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- REFMEZARFCPESH-UHFFFAOYSA-M sodium;heptane-1-sulfonate Chemical compound [Na+].CCCCCCCS([O-])(=O)=O REFMEZARFCPESH-UHFFFAOYSA-M 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 235000016709 nutrition Nutrition 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 8
- ZOIORXHNWRGPMV-UHFFFAOYSA-N acetic acid;zinc Chemical compound [Zn].CC(O)=O.CC(O)=O ZOIORXHNWRGPMV-UHFFFAOYSA-N 0.000 claims description 7
- 239000000276 potassium ferrocyanide Substances 0.000 claims description 7
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 claims description 7
- 239000004246 zinc acetate Substances 0.000 claims description 7
- 238000010829 isocratic elution Methods 0.000 claims description 5
- 238000004811 liquid chromatography Methods 0.000 claims description 5
- 230000014759 maintenance of location Effects 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000003113 dilution method Methods 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 230000000750 progressive effect Effects 0.000 claims description 3
- 239000013558 reference substance Substances 0.000 claims description 2
- 230000002441 reversible effect Effects 0.000 claims description 2
- 239000000523 sample Substances 0.000 claims 7
- 239000002245 particle Substances 0.000 claims 1
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 3
- 238000011084 recovery Methods 0.000 description 6
- 239000012086 standard solution Substances 0.000 description 5
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 3
- 238000012417 linear regression Methods 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 108060003393 Granulin Proteins 0.000 description 1
- 208000000112 Myalgia Diseases 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 208000013465 muscle pain Diseases 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000012956 testing procedure Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/64—Electrical detectors
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for measuring the content of L-glutamine in sports nutrition food, which is a high-performance liquid chromatography method. The method comprises the following steps: (1) preparing a standard stock solution and a standard working solution; (2) preparing a sample solution to be tested; (3) high performance liquid chromatography conditions; (4) establishing a standard working curve; (5) analysis of results. The method fills up the detection blank of the method for measuring the content of the L-glutamine in the sports nutrition food, is simple, convenient and quick, has good reproducibility, and is suitable for daily detection of the content of the L-glutamine in the sports nutrition food.
Description
Technical Field
The invention relates to a method for measuring the content of L-glutamine in sports nutrition food.
Background
Glutamine (Glutamine, gin) is a five-carbon amino acid, mainly exists in the form of L-glutamine, and has various physiological effects of promoting protein synthesis, improving immunity, relieving muscle pain, improving gastrointestinal barrier function, resisting fatigue and the like. In addition, studies have shown that excessive consumption of glutamine over time may cause serious adverse effects. Therefore, the invention provides a method for measuring the content of L-glutamine in sports nutrition food, which has important practical significance.
Disclosure of Invention
The invention aims to provide a method for measuring the content of L-glutamine in sports nutrition food, which is used for making up and solving the defect that no method for detecting the content of L-glutamine in the sports nutrition food exists at present.
In order to achieve the above purpose, the invention adopts the following technical scheme:
The method is a method for detecting the content of L-glutamine in sports nutrition food, and is mainly characterized by comprising the following steps of:
(1) Preparing standard stock solution and standard working solution, wherein the standard stock solution is 1.04 mg/mL standard stock solution, and the standard working solution is a series of standard working solutions with the concentration of 10.4 mug/mL, 52.0 mug/mL, 104 mug/mL, 208 mug/mL, 520 mug/mL and 1040 mug/mL, which are prepared from the standard stock solution of L-glutamine by adopting a progressive dilution method by using primary water.
(2) Preparing a sample to be tested: placing a sample to be detected in a 50 mL colorimetric tube, adding 20mL sodium heptanesulfonate solution, vortex-dissolving, adding 1 ml of 100 g/L potassium ferrocyanide solution and 1 ml of 200 g/L zinc acetate solution, fixing the volume to a scale by using the sodium heptanesulfonate solution, shaking uniformly, centrifuging at 4500r/min for 5 min, taking a proper amount of supernatant, and filtering with a 0.45 mu m filter membrane for liquid chromatography determination;
(3) The instrument is a high performance liquid chromatograph, and the detector is a photodiode array detector; the chromatographic column is a reverse C 18 column; mobile phase: the phase A is sodium heptanesulfonate solution, and the phase B is acetonitrile; isocratic elution: sodium heptanesulfonate solution (a)/acetonitrile (B) =98/2 isocratic elution; run time: 15 min; column temperature: 30 ℃; sample injection amount: 20. mu L; flow rate: 1.0 mL/min; the detection wavelength is 210 nm;
(4) Drawing a standard curve: injecting a series of standard working solutions of L-glutamine into a high performance liquid chromatograph, detecting under the chromatographic condition of the step (3), qualifying with retention time, and drawing a standard curve according to the corresponding relation between the peak area measured by each concentration and the concentration;
(5) Analysis of results: and (3) injecting the sample filtrate obtained in the step (2) into a high performance liquid chromatograph, detecting under the chromatographic condition of the step (3), measuring the peak area of the L-glutamine in the sample, qualitatively determining the retention time, quantifying according to the standard curve manufactured in the step (4), and calculating the content of the L-glutamine in the sample to be measured.
Compared with the prior art, the invention has the following outstanding advantages:
1. The invention develops a method for accurately measuring the content of L-glutamine in the sports nutritional food, and fills the blank of the technology for detecting the content of L-glutamine in the sports nutritional food.
2. According to the invention, the sodium heptanesulfonate is added into the water phase mobile phase, so that the peak shape of a target object and the separation degree of the target object from impurities are greatly improved. 3. The invention selects the potassium ferrocyanide solution and the zinc acetate solution as protein precipitants, the obtained sample extracting solution is clearer, the recovery rate is better, and meanwhile, the pollution to the instrument is avoided to a great extent.
4. The analyzer used in the invention is a high-efficiency chromatograph equipped with a photodiode array detector, and is easier to popularize and apply compared with the high-cost instruments such as a liquid chromatograph.
The invention establishes a high-efficiency chromatographic method for measuring the content of L-glutamine in the sports nutrition food. The method is simple and quick, has good reproducibility, is suitable for quantitative detection of the content of the L-glutamine in the sports nutritional food, and can provide technical support for related production enterprises, supervision departments and import and export detection departments.
Drawings
FIG. 1 is a liquid chromatography separation chart of a standard solution of L-glutamine in the sports nutritional food of the present invention.
Detailed Description
The invention is described in further detail below with reference to the drawings of the specification of the specific embodiments. The present embodiment is implemented on the premise of the present technology, and detailed embodiments and specific operation procedures are now given to illustrate the inventive aspects of the present invention, but the scope of the present invention is not limited to the following examples.
Examples
1. Reagents and materials
Unless otherwise indicated, all reagents were analytically pure and water was the secondary water specified in GB/T6682.
1.1 Acetonitrile: chromatographic purity.
1.2 Standard: L-Glutamine CAS:56-85-9, 99.2% pure, offered by Fuzhou Boke biosciences, inc.
1.3 Preparation of solution: sodium heptanesulfonate solution: 0.80g of sodium heptanesulfonate was taken, the volume was set to 1000mL with primary water, and the pH was adjusted to 2.5 with phosphoric acid. 100g/L potassium ferrocyanide solution: 10g of potassium ferrocyanide was weighed and the volume was fixed to 100mL with secondary water. 200g/L zinc acetate solution: 20g of zinc acetate was weighed and the volume was fixed to 100mL with secondary water. Preparation of standard stock solution: accurately weighing 0.1049g of L-glutamine reference substance, dissolving with primary water, and fixing the volume to 100mL to prepare a standard stock solution with the concentration of 1.04 mg/mL. Preparing a standard working solution: a series of standard working solutions having a concentration of 10.4. Mu.g/mL, 52.0. Mu.g/mL, 104. Mu.g/mL, 208. Mu.g/mL, 520. Mu.g/mL, 1040. Mu.g/mL were prepared from standard stock solutions of L-glutamine by a stepwise dilution method using primary water. All standard stock solutions and standard working solutions were kept in a refrigerator at a temperature of 4 ℃.
2. Apparatus and device
2.1 High performance liquid chromatograph: a photodiode array detector is provided.
2.2 Analytical balance (precision 0.1mg and 0.01 mg).
2.3 Vortex oscillator
2.4 High speed centrifuge
2.5 Filtration membrane: 0.45 μm.
3. Method of
3.1 Conditions for high Performance liquid chromatography
A) Chromatographic column: c 18 column (250 mm X4.6 mm,5 μm);
b) Mobile phase: sodium heptanesulfonate solution (a)/acetonitrile (B) =98/2 isocratic elution;
c) Run time: 15min;
d) Column temperature: 30 ℃;
e) Sample injection volume: 20. Mu.L;
f) Flow rate: 1.0 mL/min;
e) Detection wavelength: 210nm;
3.2 Standard Curve drawing
L-glutamine standard working solutions (1.3) were each taken and measured under the chromatographic conditions of 3.1. And (3) carrying out linear regression on the mass concentration (X, mug/mL) corresponding to the peak area (Y) of the analyte to draw a standard curve, thereby obtaining a linear regression equation.
4. Sample testing procedure
4.1 Sample pretreatment
Weighing a sample to be measured of 0.2 g to be accurate to 0.1 mg, adding 20 mL sodium heptanesulfonate solution into a 50 mL colorimetric tube, vortex-dissolving, adding 1ml of 100 g/L potassium ferrocyanide solution and 1ml of 200 g/L zinc acetate solution, fixing the volume to a scale by using the sodium heptanesulfonate solution, shaking uniformly, centrifuging at 4500 r/min for 5min, taking a proper amount of supernatant, and passing through a 0.45 mu m filter membrane for liquid chromatography determination;
4.2 sample testing
The prepared sample filtrate was chromatographed under the same chromatographic conditions, and quantified as retention time, by external standard method.
5. Representation of analysis results
The L-glutamine content in the sample was calculated as shown in the following formula (1):
Wherein:
X-L-glutamine content in grams per kilogram (g/kg) of the sample;
calculating the concentration (μg/mL) of L-glutamine in the obtained sample from the standard curve;
v-the volume of the sample is determined in milliliters (mL);
m-sample mass in grams (g).
The arithmetic mean of the results of two independent assays obtained under repetitive conditions is indicative of the result retaining three significant digits.
6. Methodology investigation, which includes linearity, detection limit, quantitative limit, recovery, precision.
6.1 Linearity, detection limit, quantitative limit: a series of mixed standard working solutions with L-glutamine concentration between 10.4 mug/mL and 1040 mug/mL were prepared, and the concentrations were subjected to linear regression with peak areas, and the linear equation and the correlation coefficient are shown in Table 1. The result shows that the L-glutamine has good linear relation within 10.4-1040 mug/mL, the correlation coefficient R is 0.9999, and the method can completely meet the detection requirement of the L-glutamine in the sports nutritional food. The detection limit and the quantitative limit of the method are shown in Table 2.
TABLE 1L-glutamine regression equation, correlation coefficient, linear Range
Y: peak area; x: mass concentration, μg/mL.
TABLE 2 detection limit and quantitative limit of L-glutamine method
6.2 Recovery rate and precision: under the optimized detection condition, a blank matrix protein powder sample is taken for carrying out a standard adding recovery rate test, the standard adding level is respectively 4.83 g/kg, 14.7 g/kg and 24.6 g/kg, and each level is repeatedly analyzed for 6 times, and the result is shown in Table 3. As shown in Table 3, the standard recovery rate of the method is 94.5% -96.8%, the relative standard deviation RSD is 0.44% -1.9%, and the result shows that the method is suitable for daily analysis and detection of the L-glutamine content in the sports nutritional food.
Table 3 protein powder sample labelling recovery experimental results (n=6)
6.3 Stability: taking L-glutamine standard solutions with three mass concentrations of 10.4 mug/mL, 104 mug/mL and 1040 mug/mL in the standard curve range, placing the L-glutamine standard solutions at normal temperature for 2h, 4h, 8 h, 10h, 12h, 16h, 20h and 24h, and examining the daily stability of the L-glutamine standard solutions. Experimental results show that the daily stability precision of the L-glutamine standard solution with the low, medium and high mass concentrations is respectively 0.82% -2.05%, 0.01% -0.38% and 0.04% -0.11%, so that the L-glutamine is stable within 24 hours, and the experiment is facilitated.
Finally, it is noted that the above-mentioned embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (5)
1. A method for determining the L-glutamine content of a sports nutritional food product, said method comprising:
(1) Preparing standard stock solution and standard working solution, wherein the standard stock solution is 1.04mg/mL standard stock solution, and the standard working solution is a series of standard working solutions with the concentration of 10.4 mug/mL, 52.0 mug/mL, 104 mug/mL, 208 mug/mL, 520 mug/mL and 1040 mug/mL, which are prepared from the standard stock solution of L-glutamine by adopting a progressive dilution method by using primary water;
(2) Preparing a sample to be tested: placing a sample to be detected in a 50 mL colorimetric tube, adding 20mL sodium heptanesulfonate solution, vortex-dissolving, adding 1ml of 100 g/L potassium ferrocyanide solution and 1ml of 200 g/L zinc acetate solution, fixing the volume to a scale by using the sodium heptanesulfonate solution, shaking uniformly, centrifuging at 4500 r/min for 5min, taking a proper amount of supernatant, and passing through a 0.45 mu m water phase filter membrane for liquid chromatography determination;
(3) The instrument is a high performance liquid chromatograph, and the detector is a photodiode array detector; the chromatographic column is a reverse C 18 column; mobile phase: the phase A is sodium heptanesulfonate solution, and the phase B is acetonitrile; isocratic elution: sodium heptanesulfonate solution (a)/acetonitrile (B) =98/2 isocratic elution; run time: 15min; column temperature: 30 ℃; sample injection amount: 20. Mu.L; flow rate: 1.0 mL/min; the detection wavelength is 210nm;
(4) Drawing a standard curve: injecting a series of standard working solutions of L-glutamine into a high performance liquid chromatograph, detecting under the chromatographic condition of the step (3), qualifying with retention time, and drawing a standard curve according to the corresponding relation between the peak area measured by each concentration and the concentration;
(5) Analysis of results: and (3) injecting the sample filtrate obtained in the step (2) into a high performance liquid chromatograph, detecting under the chromatographic condition of the step (3), measuring the peak area of the L-glutamine in the sample, qualitatively determining the retention time, quantifying according to the standard curve manufactured in the step (4), and calculating the content of the L-glutamine in the sample to be measured.
2. The method for measuring the L-glutamine content in a sports nutritional food according to claim 1, wherein the preparation of the standard stock solution in (1) specifically comprises the steps of: accurately weighing 0.1049g of L-glutamine reference substance, dissolving with primary water, and fixing the volume to 100mL to prepare standard stock solution with the concentration of 1.04 mg/mL; (1) The preparation of the standard working solution comprises the following specific steps: a series of standard working solutions with the concentration of 10.4 mug/mL, 52.0 mug/mL, 104 mug/mL, 208 mug/mL, 520 mug/mL and 1040 mug/mL are prepared from standard stock solution of L-glutamine by adopting a progressive dilution method by using primary water, and all the standard stock solution and the standard working solution are stored at the temperature of 4 ℃ of a refrigerator.
3. The method for measuring the content of L-glutamine in a sports nutritional food according to claim 1, wherein the preparation of the sample solution to be measured in (2) specifically comprises the steps of: weighing 0.2g of a sample to be measured to be 0.1mg accurately, adding 20 mL sodium heptanesulfonate solution into a 50 mL colorimetric tube, vortex-dissolving, adding 1 ml of 100 g/L potassium ferrocyanide solution and 1 ml of 200 g/L zinc acetate solution, fixing the volume to a scale by using the sodium heptanesulfonate solution, shaking uniformly, centrifuging at 4500 r/min for 5min, taking a proper amount of supernatant, filtering with a 0.45 mu m filter membrane, and waiting for liquid chromatography to determine.
4. The method for measuring the L-glutamine content in a sports nutritional food according to claim 1, wherein said reversed-phase C 18 column in (3) has a specification of 250mm by 4.6mm inside diameter, and a particle diameter of 5. Mu.m.
5. The method for measuring the L-glutamine content in a sports nutritional food according to claim 1, wherein the standard curve correlation coefficient in (4) is not less than 0.999.
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